An integrated workflow for phosphopeptide identification in natural killer cells (NK-92MI) and their targets (MDA-MB-231) during immunological synapse formation

Summary Here, we present a protocol to identify and quantify phosphopeptides during the dynamic formation of an immunological synapse. We describe steps for mixing isotope-labeled immune and target cells, the stabilization of cell-to-cell conjugates by cross-linking, and their isolation by fluorescence-activated cell sorting. We detail the isolation of phosphopeptides by phosphopeptide enrichment and their subsequent measurement by mass spectrometry. Finally, we describe the analysis of the resulting data to separate cell-specific phosphopeptides using the isotope label and label-free quantification.

1. Prepare buffers following the recipes in the materials and equipment. 2. All reagents can be stored at room temperature for 2 months, except the ones indicated by the manufacturer: a. Trypsin should be prepared fresh at 0.25 mg/mL in 50 mM ammonium bicarbonate buffer. b. Reduction buffer and Alkylation buffer should be prepared fresh.
Note: Alkylation buffer should be kept in the dark.
Note: The protocol below describes the sample preparation for approximately 25 samples. For a larger number of samples, the volumes need to be adjusted.  Note: MDA-MB-231 were purchased from ATCC and subsequently genetically modified to stably express a fluorescent actin reporter for our specific research interest (Al Absi et al. 4 ).

MATERIALS AND EQUIPMENT Buffers resource table
Note: All reagents are mixed and stored at 4 C for up to 1 month, except for amino acids that are stored at À20 C and added just before using the medium. Note: All reagents are mixed and stored at 4 C for up to 1 month.

Mass spectrometry parameters
This protocol is specifically set up for phosphopeptide analysis in low concentration. The conditions are optimized for Ultimate 3000 UHPLC system (DIONEX) coupled to Q Exactive HF mass spectrometer (ThermoFisher Scientific), using the Nanospray Flex ion source at 275 C (ThermoFisher Scientific). The MS was calibrated following the manufacturer instructions. The MS was operated in data-dependent mode with the following parameters:

UHPLC parameters
The UHPLC setup was injecting in two-column mode: the loading pump injects the sample in the loop in ''microliter pickup mode'', and sample is transferred to the Acclaim PepMap RSLC 75mm 3 2 cm nanoViper (pepTrap) for 8 min at 5 mL/min. After 8 min, the column-oven valve switches to put both pepTrap and the Acclaim PepMap RSLC 75mm 3 15 cm nanoViper online to start the gradient. The parameters are described below:

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Alternatives: Other LC-MS/MS setups can be used for the MS analysis, as long as they can analyze SILAC-labeled samples. 10,14 The conditions must be adjusted according to the instrumentation, and the final concentrations and volumes of the samples have to be adjusted. Other SDS-PAGE systems can be used for sample cleaning and fractionation, but it is important to keep the protein amount at 25 mg for further phosphopeptide enrichment and mass spectrometry analysis.

STEP-BY-STEP METHOD DETAILS
Production of SILAC-labeled NK-target cell conjugates Cell labeling in SILAC medium This section describes the labeling of the cells in SILAC media. NK-92MI cells are cultured in lightlabeled and the MDA-MB-231 cells in heavy-labeled SILAC media. 10 Duration of cell culture in SILAC media depends on two parameters: the labeling efficiency of proteins and the quantity of cells needed to form a sufficient number of cell-to-cell conjugates. The labeling efficiency for each cell type is evaluated after five serial cell passages and should exceed 95% labeling. If the labeling efficiency is not high enough, the cells should be cultured over an extended period of time until the threshold of 95% is reached.
To optimize cell culture, the size of cell culture flasks is adjusted to the number of cells necessary for coculture.
1. NK-92MI cell culture. a. NK-92MI are cultured in 30 mL RPMI SILAC medium in 75 cm 2 flasks and used after 5 passages if the labeling efficiency of the cells exceeds 95%.
Note: add the isotope-labeled amino acids freshly.
b. Every 2-3 days, cells are counted to adjust the concentration at 3*10 5 cells/mL (1 passage). c. To proceed with step 2: 10 5 -10 6 cells are sufficient for a preliminary test. d. To proceed with step 3: 3*10 6 are sufficient. e. To proceed with step 4: 35*10 6 NK-92MI cells are required. CRITICAL: It is necessary to perform a labeling efficiency test on heavy K and R to confirm that >95% of the proteins are heavy-labeled before proceeding to the next step. In case the labeling efficiency is < 95% go to problem 1. CRITICAL: It is necessary to cultivate both cell lines in SILAC media, even if light amino acids are used, to ensure that no excess amino acids are carried over into the next steps.

Determination of the labeling efficiency
Timing: 2 days (for steps 3 to 22) In this section, the degree of incorporation of the isotope-labeled amino acids is determined by a mass spectrometric measurement.
3. Take an aliquot of the heavy labeled cells (cell culture section). A total of 10 5 -10 6 cells are sufficient for the preliminary test. 4. Lyse cells with 100 mL of lysis buffer on ice.
Note: prepare Lysis buffer freshly before use. 5. Vortex for 10 s at maximum setting. 6. Incubate cells at 4 C for 15 min to allow the mixture to sit. 7. Vortex for 10 s at maximum setting. 8. Centrifuge in a tabletop bench centrifuge at 4 C at maximum speed (16,000 g) to remove cell debris. 9. Transfer supernatant to a 2 mL Eppendorf tube. 10. Estimate protein concentration with BCA assay. The protein concentration should be between 0.5-2 mg/mL. 11. Add 5 mL of reduction buffer and incubate for 30 min at RT. 12. Add 5 mL of alkylation buffer and incubate for 30 min at RT in the dark. 13. Dilute sample 1:4 with dilution buffer to decrease urea concentration below 2 M. 14. Add trypsin buffer (substrate ratio of 1:50) for overnight at RT. 15. Add 10 mL of stop buffer. 16. Perform step 5 subsection, ''digest clean up'' to clean the peptides. 17. Reconstitute the samples in ST buffer B to obtain approximately 1 mg/mL. 18. Inject 1 mg of material into the LC-MS/MS with the parameters described in the mass spectrometry resource parameters section. 19. Analyze the data with MaxQuant following the protocol described in section: automated interpretation of the mass spectra using MaxQuant, with exceptions: 20. Under ''variable modifications'', add Pro6 (determine arginine-to-proline conversion). 21. Select multiplicity 2 and mark the labels as Lys8 and Arg10. Do not select the ''re-quantify'' option (described in 10 ). 22. The labeling efficiency should be calculated based on the non-normalized SILAC ratios and calculated for K and R separately.
Note: Determine the incorporation rate as: Complete labeling is considered when the incorporation rate is higher than 95%. Heavy proline (a side-product of arginine labeling) should not exceed 1%.

Cell-to-cell conjugation efficiency test
Timing: 3 h (for step 23)

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The following section describes the formation and subsequent chemical cross-linking of cell-to-cell conjugates. For the isolation of the conjugates, natural killer cells and target cells are marked by two different fluorescent labels. Labeling can be achieved using immunofluorescence staining or expression of recombinant fluorescent proteins. Here, NK-92MI cells are labeled using a PE-Cy7 conjugated anti-CD56 antibody prior to conjugate formation, while MDA-MB-231 cells are identified by mEmerald-actin-fusion. 4 Both cell types are co-incubated for 10 min to allow the formation of cell-to-cell conjugates and fixed with paraformaldehyde (PFA). Double-labeled conjugates are identified and isolated using FACS.
23. Calculate the number of cells required to reach the critical amount of cell-to-cell conjugates necessary for subsequent mass spectrometry analysis.
Note: An example is given below (Figure 2) following the method described in step 4 and step 5 with a 1:10 cell amount. In our experiment, we use the BD FACS Aria (Becton Dickinson Bioscience) for FACS analysis and sorting. Other instruments can be used, but the parameters have to be adjusted accordingly.

Conjugate formation and chemical cross-linking
Timing: 3 h (for steps 24 to 28) A minimum of 25 mg of cell-to-cell conjugates (approximately 10 6 conjugated cells) is necessary for mass spectrometry analysis. To calculate the number of NK-92MI cells and MDA-MB-231 cells that should be mixed in order to reach the critical number of cell-to-cell conjugates, both the NK-92MI:MDA-MB-231 cell ratio (i.e., 3:1) and the conjugation rate at this given ratio (i.e., 5%-10% of the total cell number) should be taken into account. It is important to test the cell-to-cell conjugation efficiency test first (conditions described in step 3) for further mass spectrometry experiments.
24. Staining of the NK-92 MI cells. a. Count NK-92MI cells using a Neubauer chamber with trypan blue solution (follow the manufacturer's instructions). b. Transfer 35*10 6 NK-92MI cells in a 15 mL falcon tube. c. Centrifuge at 300 3 g for 5 min. Note: The required quantities should be recalculated according to the number of cells.
Note: To reduce the risk of interfering with the subsequent steps, in particular mass spectrometric analysis, and recover unlabeled living cells, we recommend using (fixable) cell membrane impermeant viability dyes that specifically stain the nucleus of dead cells, such as Live-or-Dye NucFixä Red. Note: PFA must be diluted in PBS/Mg/Ca to keep conjugates maintained.
Note: The required quantities should be recalculated according to the number of cells.

Cell sorting
Timing: 1 h (for step 29) Timing: 1 day (for step 30) Step 5 describes the key steps for data acquisition and/or cell sorting. For further details on flow cytometry, the reader is referred to Cossarizza   Proteins are separated on a pre-casted SDS gel into three fractions to decrease the complexity of the sample. A lower complexity will increase the number of identified phosphopeptides. The SDS-gel

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functions two-fold: first, the heating of the samples reverses the PFA cross-links, and second, it removes substances that are interfering with the mass spectrometric analysis. Proteins are digested into peptides in the SDS-gel, and the resulting peptides are extracted. The contained phosphopeptides are enriched using metallo-chelate chromatography before being analyzed by mass spectrometry.
Note: The starting material should not be below 0.35*10 6 cell-to-cell conjugates, which approximately corresponds to 25 mg per sample. CAUTION: The coomassie brilliant blue dye from the gel pieces should vanish for optimal digestion. If the coomassie brilliant blue dye remains, add 3 more washes (step 33c).
CAUTION: The gel pieces should be wet in all the procedures; otherwise, the reproducibility of digestion will be compromised.
CRITICAL: If the gel pieces absorb all the buffer, add 50 mL digestion buffer. In case the digestion buffer is increased, the stop buffer should also be adjusted to a final concentration of 0.1% formic acid. Pause point: The purified peptides can be stored one month at -80 C.

Phosphopeptide enrichment
Timing: 1 h (for steps 35 to 36) Phosphopeptides are usually low abundant and are difficult to be detected in the mass spectrometer. Therefore, the phosphopeptides need to be enriched using a specific metallo-chelate chromatography.
35. Reconstitute the samples in the starting buffer and perform the phosphopeptide enrichment as specified in High-Select Fe-NTA Phosphopeptide Enrichment Kit.
Note: this step enables the enrichment of phosphorylated peptides from protein extracts using iron-chelate resin in spin columns. The capacity of the columns ranges from 0.5 to 5 mg of total digested protein sample and can enrich up to 150 mg phosphopeptides with high selectivity.
36. In the last step of the kit protocol, resuspend the material in 12 mL of 0.1% formic acid to inject into the mass spectrometer.
CRITICAL: check the color of the Fe 3+ -NTA beads. The color should be black. If the beads turn yellow, the beads are oxidized and will not work.
Note: if the analysis of the sample does not show enough phosphopeptides, See the troubleshooting of the High-Select Fe-NTA Phosphopeptide Enrichment Kit.
Pause point: The eluted peptides can be stored at -80 C for one month.

Mass spectrometry analysis (MS)
Timing: 1 day (dependent on the computer hardware available) (for step 37) The recorded mass spectrometric data has to be matched to peptides and proteins. For this, we use the MaxQuant software package. MaxQuant demands significant computing resources. In our case, we used a 72-core computer to analyze the data. Using a smaller computer will prolong the time needed for the analysis. Note: the parameters that are described below are a guide for a specific mass spectrometer, but others could be used to perform MS analysis.
Optional: The amount of phosphopeptides to inject could differ depending on the peptide abundance, the mass spectrometer, and the nanoHPLC system. Those conditions have to be adjusted injecting the corresponding volume into LC-MS/MS. For a Q Exactive HF (Thermo Scientific) 0.1 mg of material is enough to generate a robust dataset (around 2,500 peptides per measurement, which corresponds to approximately 2 3 10 9 of maximum total ion count (TIC)).
To interpret the mass spectrometry data, use the MaxQuant software package (here, we use version 1.6.17.0) with the following search parameters. The rest of the MaxQuant parameters should be maintained as default for optimal data processing. 12

Bioinformatic analysis and data interpretation
Timing: 2 days (for steps 38 to 46) In this step, the data is analyzed using statistical tools ( Figure 5).
The MaxQuant search engine generates several files while processing the raw data from ThermoFisher instruments. To extract the files needed for the analysis: go to the combined folder, and then to the txt folder. The samples are analyzed by R-based programming or Perseus. 18,19 Perseus is convenient for filtering data (114-116). For the quantification (117-121) we recommend R-based scripting to analyze the data. An example of the data analysis is shown in Table S1. are considered as valid phosphorylation (positive) or dephosphorylation (negative). The rest of the phosphosites will be discarded as the signal is not sufficient to distinguish from background signals.
43. The abundance changes in the phosphosites also depend on the differences in protein abundance between those two samples. We monitor this difference by Z-scoring of the LFQ Intensity H in proteingroups.txt file. 44. log 2 LFQ Intensity H MDA À NK LFQ Intensity H MDA < 1 will not be considered as protein abundance change.

log 2
LFQ Intensity H MDA À NK LFQ Intensity H MDA > 1 Proteins that include phosphosites changing in the same direction will be discarded. 46. The SILAC label compares the protein abundance between MDA-MB-231 and NK92-MI in conjugates.
Note: the SILAC pairs in NK92-MI will be considered as an artifact (only L label in the sample), but the SILAC pair in MDAs (H label) will be considered to perform a quality control for labeling efficiency.

EXPECTED OUTCOMES
The provided datasets originate from an analysis using the described method. The data analysis of the dataset leads to the identification of 1197 phosphoproteins and 2800 phosphopeptides, which The two different SILAC isotope markers are used as different channels and compared to the single cells using LFQ quantification to identify cell type-specific events.

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can be assigned to different cell types of cell-to-cell conjugates. Experiments using other cell types should identify phosphoproteins and -peptides in the same range.

LIMITATIONS
The analysis is based on SILAC-labeling to distinguish where the phosphorylated peptides originate. Thus, the method can only be used on cells that can be SILAC-labeled. The number of cell-to-cell conjugates formed by the two cell populations can vary depending on the cell type and might be a limiting factor for some cell type combinations. If one cell type can form homotypic cell-to-cell conjugates, the strategy for FACS needs to be adjusted and fitted to select conjugated cells from single cells. A minimal number of phosphorylated peptides are necessary for the mass spectrometric analysis to get sufficient identifications. Depending on the conjugation efficiency, the amounts have to be adjusted. In general, more replicates will lead to a more robust coverage of the phospho-proteome.

Problem 1
The labeling efficiency of the cells is <95% due to problems with the dialyzed serum or the number of passages is not enough to incorporate all the labeled amino acids (steps 1 and 2).

Potential solution
Wash cells with PBS and perform 4 more passages with a fresh dialyzed serum before a new test. Check if the SILAC media does contain the right amount of SILAC amino acids for labeling.

Problem 2
Centrifugation and pipetting before the fixation step could alter cell-to-cell conjugates (steps 26-28). The sheer forces within the pipette can separate cells from each other.

Potential solution
Pipet gently and limit washing steps by pre-labeling cells for viability before NK92-MI cells and MDA-MB-231 conjugation. The 10 min co-culture time is insufficient to induce strong NK92-MI cell cytotoxic activity, specifically with MDA-MB-231 characterized by their resistance to NK92-MI cell attack.

Problem 3
The sorting efficiency (for both NK92-MI cell-MDA-MB-231 cell conjugates and unmixed cells), as defined by the number of positive events sorted divided by the number of positive events detected, has to be larger than 30%.

Potential solution
The sorting efficiency depends on several parameters that could be modified to improve it: Aggregates formed by cell clustering can be limited by a filtration step with 100 cell strainers before the sorting. The cell concentration per mL is important to limit the exclusion of positive events because of too close proximity to a negative event. The cell suspension could be diluted to limit this phenomenon. A first sorting can improve the percentage of positive events in the cell suspension based on yield and not purity. The machine's settings on yield mode allow the recovery of all positive events, despite the proximity to negative events that could be included in the sorted drop. The recovered cell suspension is enriched in positive events and can be sorted again on purity mode. The machine will exclude all drops that could contain any non-target events.

Problem 4
There is no clean separation between the different bands in the SDS-PAGE.

Potential solution
Generally, this is due to the high concentration of PFA or high amounts of DNA in the sample. Add 8 mL of 10% SDS instead of 5 mL and boil for 15 min at 95 C. Use sonication to break down the DNA or add 5 mL of Benzonase for 20 min at RT.

Problem 5
The sample is not homogeneous, and some of the samples is not passing through the gel or is trapped in the gel pocket.

Potential solution
The sample was not soluble because of the cell debris. Perform an ultra-centrifugation at 100,000 g for 20 min after boiling the sample, recovering the non-solid phase (upper phase) before loading it onto the SDS-PAGE.

RESOURCE AVAILABILITY
Lead contact Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Gunnar Dittmar (gunnar.dittmar@lih.lu).

Materials availability
This study did not generate new unique reagents.

Data and code availability
The mass spectrometric dataset generated during this study are available at MASSIVE proteomeXchange: MSV000090645. ftp://massive.ucsd.edu/MSV000090645/